Split Recombinase System for Marker-Free Fungal Strain Construction

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Solution Overview

Problem

The limited availability of selectable markers for filamentous fungi complicates the construction of new cell lines, and the use of antibiotic resistance markers is risky due to potential biosphere contamination risks, necessitating the development of methods for efficient recombination at target loci without leaving residual markers.

Innovation Solution

A method involving the use of nucleic acids capable of homologous and site-specific recombination to insert and remove sequences at target loci, utilizing a split-recombinase approach to facilitate marker removal and avoid unwanted recombinase activity, allowing for efficient integration and out-recombination of markers in fungal cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If antibiotic resistance markers are used for strain construction, then selection efficiency is improved, but biosphere contamination risk increases

Engineering Contradiction:

Improvestrain construction efficiencyVSAvoidbiosphere contamination risk

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The invention extracts and removes the antibiotic resistance marker from the final strain product. The marker is used temporarily during strain construction but is then excised using site-specific recombination systems (Cre/loxP, FLP/frt, or Dat/att), leaving no residual antibiotic resistance genes in the production strain, thus eliminating biosphere contamination risk while maintaining selection efficiency during the construction process

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The marker gene is discarded after serving its selection purpose. The system allows the marker to be introduced for efficient selection of transformed cells, then systematically removed through recombination events, recovering a marker-free strain suitable for commercial production without antibiotic resistance concerns

Inventive Principle:

Principle #34Discarding and recovering

2Adaptability or versatility

If multiple selectable markers are used for sequential strain modification, then genetic engineering flexibility is improved, but marker availability is depleted

Engineering Contradiction:

Improvestrain modification flexibilityVSAvoidnumber of available markers

Core Design Contradiction:

Adaptability or versatilityVSQuantity of substance

Solution Approach 1:

The same selectable marker can be reused multiple times because it is completely removed after each strain modification step. The marker is introduced for selection, then excised via site-specific recombination, allowing the same marker gene to be recovered and used in subsequent strain construction projects, eliminating the need to deplete the limited marker repertoire

Inventive Principle:

Principle #34Discarding and recovering

Solution Approach 2:

The strain construction process is segmented into distinct phases: marker introduction for selection, marker removal via recombination, and marker recovery for reuse. This segmentation allows the marker to serve its purpose temporarily and then be restored to the available pool, enabling unlimited sequential modifications with a limited set of markers

Inventive Principle:

Principle #1Segmentation

3Manufacturing precision

If site-specific recombination sites are introduced, then marker removal precision is improved, but genome complexity increases

Engineering Contradiction:

Improvemarker removal accuracyVSAvoidgenome structure complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

Site-specific recombination sites (loxP, frt, or att) serve as intermediary elements that facilitate precise marker removal. These short DNA sequences act as recognition targets for recombinase enzymes, enabling accurate excision of markers at specific locations without requiring complex genomic rearrangements or leaving large residual sequences, thus achieving high precision with minimal genome complexity increase

Inventive Principle:

Principle #24Intermediary (Mediator)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Enables efficient and marker-free strain construction in fungi, reducing the risk of biosphere contamination and simplifying the process by allowing for one-step transformation and efficient out-recombination of selection markers, thereby enhancing the safety and productivity of fungal strain development.

Implementation Method 1

two or more site-specific recombination sites; and (c) a sequence encoding a recombinase which recognizes the site-specific recombination sites

Methodology Applied

Scientific EffectSite-specific recombination:

Implementation Method 2

the two or more nucleic acids are capable of homologous recombination with each other so as to give rise to a single nucleic acid

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentUS10793850B2Recombination system

Publication Date: 2020.10.06 DSM IP ASSETS BV

AI summary

The present invention relates to a method for carrying out recombination at a target locus.